Protease-activated receptors (PARs) are G-protein coupled receptors (GPCRs) with a unique mode of activation involving the cleavage of the N-terminus of the receptor by various proteases. Among other activities, these receptors play a vital role in the activation of platelet cells in hemostasis, as well as regulating the immune response in endothelial and immune cells. The pleiotropic effects mediated by PARs are governed by signaling pathways involving different G-proteins and B-arrestins, and evidence has accumulated demonstrating that the balance of signaling for PARs, and GPCRs in general, depends on the nature of their ligands as well as the physical environment of the receptor. Evidence also suggests that in certain contexts some PAR-mediated signals may be beneficial, and others harmful. This proposal outlines the design and development of two classes of novel pharmacological tools for PAR-mediated signaling, as well as the potential application of these tools to the treatment of cardiovascular disease and sepsis. First, analogs of the small molecule PAR1 biased antagonist ML161 will be prepared to study structure-activity relationships (SARs) and structure-function relationships that will guide the understanding of biased signaling of PAR1, an important target for a new class of anti-platelet drugs for cardiovascular disease. Analogs will be prepared with an additional focus on the improvement of physiochemical properties to support future in vivo studies of these compounds that may offer additional cytoprotective effects not observed with orthosteric PAR1 antagonists. The mode of action of these compounds, termed parmodulins, will also be studied via photoaffinity experiments using novel bifunctional probes to guide the identification of a specific allosteric binding site. In our second approach, we will study the relevance of PAR1-PAR2 receptor complexes to the treatment of sepsis, the pathological immune response to severe infection. Novel heterobivalent ligands targeting these complexes will be prepared and tested for their ability to selectively target such co-complexes in endothelium exposed to LPS, an in vitro model for sepsis. These compounds offer for the first time the possibility of tissue- and context-selective PAR ligands with promise for the treatment of sepsis and other PAR-associated pathologies.